paul j. martin, m.d. division of clinical research,...
TRANSCRIPT
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0B0BBiology of Chronic Graft-versus-host Disease: Implications for a Future Therapeutic
Approach
Key words: graft-versus-host disease, hematopoietic cell transplantation,
Paul J. Martin, M.D.
Division of Clinical Research, Fred Hutchinson Cancer Research Center, Department of
Medicine, University of Washington
Correspondence: Paul J. Martin, M.D.
Fred Hutchinson Cancer Research Center, D2-100
P.O. Box 19024
Seattle, WA 98109-1024
Running title: Biology of Chronic Graft-versus-host Disease
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Abstract
Hematopoietic cell transplantation (HCT) is frequently complicated by graft-versus-host
disease (GVHD). During the past three decades, experimental studies and clinical
observations have elucidated the pathophysiology of acute GVHD, but the biology of
chronic GVHD is much less well understood. Recommendations of the NIH Consensus
Development Project on Criteria for Clinical Trials in Chronic GVHD have begun to
standardize the diagnosis and clinical assessment of the disease. These criteria have
emphasized the importance of qualitative differences, as opposed to time of onset after
HCT, in making the distinction between acute and chronic GVHD. Experimental studies
have generated at least four theories to explain the pathophysiology of chronic GVHD.
These theories include 1) thymic damage and defective negative selection of T cells
generated from marrow progenitors after HCT, 2) aberrant production of transforming
growth factor-β, 3) auto-antibody production, and 4) deficiency of T-regulatory cells.
Recent studies in humans have corroborated a possible role for each of these
mechanisms in humans. No animal model fully replicates all of the features of chronic
GVHD in humans, and it appears likely that multiple biological mechanisms account for the
diverse features the disease. Chronic GVHD may represent a “syndrome” with diverse
causes among individual patients. In the future, it might become possible to tailor specific
therapeutic interventions for patients as individually needed for each distinct
pathophysiologic mechanism involved in development of the disease.
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Clinical presentation and significance of chronic GVHD
Chronic GVHD is a pleiomorphic syndrome with onset generally occurring between 3 and
24 months after allogeneic hematopoietic cell transplantation (Table 1).1 The highly
variable clinical manifestations of chronic GVHD frequently involve the skin, liver, eyes,
mouth, upper respiratory tract, esophagus, and less frequently involve serosal surfaces,
lower gastrointestinal tract, female genitalia, and fascia.2 The biological mechanisms
leading to chronic GVHD are not as well understood as those leading to acute GVHD.
Although acute GVHD has been recognized as a risk factor for chronic GVHD, not all
cases of acute GVHD evolve into chronic GVHD, and chronic GVHD can develop in the
absence of any prior overt acute GVHD. In the skin, the initial phase of chronic GVHD is
characterized by an intense mononuclear inflammatory infiltrate with destructive changes
at the dermal-epidermal junction, accompanied by irregular acanthosis, hyperkeratosis or
atrophy, progressing to dermal fibrosis and sclerosis.3 Other hallmarks include destruction
of tubuloalveolar glands and ducts in the skin, salivary and lacrimal glands and respiratory
epithelium, and destruction of bile ducts in the liver.
Immune-mediated mechanisms of fibrosis
Insight regarding the pathophysiology and immunobiology of chronic GVHD is limited. A
wide variety of experimental models have indicated an association between type-2
polarized immune responses and the development of fibrosis,4 and donor type 2 immune
responses are required for induction of skin GVHD in mice.5 Complement factor 5 (C5) has
been identified as a quantitative trait that modifies liver fibrosis in mice and humans,6 and
C5b-9 complexes are deposited in the skin, liver, lung and kidney in mice with GVHD.7 C3
is deposited at the dermal-epidermal junction in humans with chronic GVHD,8 but
deposition of C5b-9 complexes has not been described.
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Animal models of chronic GVHD
At least four different hypotheses regarding the pathogenesis of chronic GVHD have
emerged from studies with animal models. One hypothesis posits that chronic GVHD
results from thymic damage caused by acute GVHD, resulting in failure to delete nascent
T cells that recognize antigens on donor or recipient cells. A second hypothesis implicates
a central role for TGF-β in the pathogenesis of the disease. A third hypothesis implicates B
cells and antibody-mediated mechanisms in certain manifestations of the disease. Lastly,
deficiency in the numbers or function of T-regulatory cells might contribute to the
development of chronic GVHD.
The literature is confusing because the term “chronic GVHD” has been used to
describe a syndrome of antibody-mediated glomerulonephritis that occurs when recipient
B cells are activated by donor CD4 cells after transplantation of spleen cells from certain
parental strains into non-irradiated F1 mice. The resulting nephrotic syndrome is more
characteristic of lupus nephritis as opposed to chronic GVHD, although case reports have
occasionally described nephrotic syndrome in patients with chronic GVHD.
Failure of negative selection in the thymus. Cutaneous changes of acute and chronic
GVHD occur in H-2b MHC-identical transplants between LP and C57BL/6 (B6) mice.
Parkman9 showed that clones from B6 recipients with chronic GVHD were all CD4+ and all
showed IL-2-dependent proliferative responses specific for MHC class II I-Ab antigens
expressed by both the donor and recipient. The observation that CD4+ clones from
recipients with chronic GVHD showed specificity for I-Ab suggested that these cells had
emerged from marrow progenitors that escaped negative selection in the thymus, and the
observation that similar clones could be detected in mice with acute GVHD suggested that
the processes responsible for generating such autoimmune clones begin early after
transplantation.
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Acute GVHD causes severe histopathological damage in the thymus, including injury
to medullary epithelial cells, effacement of the corticomedullary junction, disappearance of
Hassal’s corpuscles and depletion of CD4+CD8+ cells.10 In the thymus, developing T cells
that express receptors with high affinity for peptide-MHC complexes or “self”-antigens on
adjacent cells are deleted. This process of negative selection occurs in the medulla and is
mediated by marrow-derived dendritic cells and thymic medullary epithelial cells. Negative
thymic selection among T cells developing in mice with GVHD is impaired,11 and the T
cells developing in mice with GVHD are pathogenic. Zhang et al.12 showed that dendritic
cells were depleted in the thymus of B6 mice with acute GVHD caused by donor C3H.SW
CD8 cells. The resulting thymic damage allowed the development of CD4 cells that
responded vigorously to recipient B6 alloantigens. These CD4 cells caused chronic GVHD
when they were adoptively transferred into irradiated secondary B6 recipients.
Administration of keratinocyte growth factor (KGF) at the time of the transplant enhanced
reconstitution of dendritic cells in the thymus, and the CD4 cells that emerged from KGF-
treated recipients did not cause GVHD in secondary B6 recipients.12
Zhang et al.12 also showed that donor-derived C3H.SW CD4 cells from B6 recipients
with GVHD caused acute hepatic and intestinal GVHD when they were adoptively
transferred into irradiated secondary C3H.SW recipients. Further evidence for
“autoimmune” recognition among T cells developing in mice with GVHD was reported by
Tivol et al.13 in experiments where adoptively transferred spleen cells from B6 → BALB/c
chimeras with GVHD were adoptively transferred into nonirradiated secondary
immunodeficient B6 or BALB/c recipients. Cells from the chimeras caused colitis in
secondary B6 recipients but not in BALB/c recipients. In a different approach, several
groups have shown that transplantation of MHC-class II-deficient marrow into wild-type
recipients of the same strain causes autoimmune damage in the skin,14 liver and
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intestines.15 Thymectomy prevented the disease, and adoptive transfer of CD4+ cells
caused acute GVHD in irradiated secondary recipients of the same strain.
In a variation of the same approach, Sakoda et al.16 found that irradiated C3H (H-2k)
recipients reconstituted with T cell-depleted marrow from MHC-class II-deficient B6 (H-2b)
donors developed a disease with clinical and histopathological features characteristic of
chronic GVHD, including epidermal atrophy, follicular dropout, fat loss, dermal fibrosis, bile
duct loss, with inflammation, atrophy and fibrosis of acinar tissue in the salivary glands.
Thymectomy prevented the disease, and the chimeric CD4 cells proliferated in response to
donor-type B6 antigen-presenting cells (APCs) but not in response to recipient-type C3H
APCs. Adoptive transfer of chimeric CD4 cells in the presence of B6 APCs caused chronic
GVHD in irradiated secondary C3H recipients. In irradiated secondary B6 recipients,
however, the adoptively transferred CD4 cells caused acute GVHD.
These results are consistent with those of an earlier study suggesting that CD4 cells
cause acute GVHD when they recognize antigens expressed by recipient epithelial tissues
but cause chronic GVHD when they recognize antigens on marrow-derived cells but not on
epithelial tissues.17 In these models, the acute or chronic nature of GVHD appears to be
dictated respectively by the presence or absence of the recognized antigens on epithelial
cells of the recipient. Similar experiments have been done with B6 donors and MHC-
mismatched BALB/c or MHC-matched BALB.B recipients. Irradiated secondary BALB/c
recipients developed acute GVHD,18 while secondary BALB.B recipients developed
chronic GVHD19 after adoptive transfer of CD4 cells from chimeric donors with GVHD.
Hence, an absence of the recognized antigens on recipient epithelial cells does not
entirely explain the development of chronic GVHD.
Further work is needed to define the B6 antigens that stimulate proliferation of donor
CD4 cells in the model described by Sakoda et al.16 In principle, the CD4 cells that develop
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in the chimeras are positively selected by thymic cortical epithelial cells of the C3H
recipient and negatively selected by MHC class I-positive B6 APCs and by recipient C3H
epithelial cells in the thymic medulla. Although the donor-derived CD4 cells that escape
MHC-class II-specific negative selection respond to wild type B6 APCs in vitro, the B6-
derived APCs in the primary recipients do not express MHC-class II molecules and would
not be expected to stimulate donor-derived CD4 cells in vivo. Evidence from at least one
study has suggested that the CD4 cells escaping negative selection in the thymus respond
to MHC class I antigens.20
Taken together, the experiments with mice demonstrate that acute GVHD impairs
negative selection of T cells in the thymus and that CD4 cells recognizing donor or
recipient alloantigens can cause a syndrome with remarkable similarity to chronic GVHD.
Further work is needed to determine whether these observations have relevance for
chronic GVHD in humans. As reported by Tsoi et al.21 in 1980, donor-derived T cells from
patients showed proliferative responses after stimulation with cells from HLA-identical
sibling recipients in 14 of 22 (64%) cases with chronic GVHD and in only 1 of 12 cases
without chronic GVHD (Table 2). Responses after stimulation with recipient cells could not
be explained by progeny of mature T cells in the graft, since T cells from patients with
acute GVHD did not respond after stimulation with recipient cells. The presence of
alloreactive T cells in these patients suggests an impairment of negative selection by
thymic medullary epithelial cells of the recipient. Responses after stimulation with donor
cells were observed 7 of 22 cases (32%) without chronic GVHD and in only 1 of 12 cases
without chronic GVHD. In all but one of these 8 cases, responses were also observed after
stimulation with recipient cells. These results suggest that the primary defect associated
with chronic GVHD in humans is an impairment of negative selection mediated by
medullary thymic epithelial cells, with or without concomitant impairment of negative
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selection mediated by donor-derived dendritic cells. It seems unlikely that this mechanism
can account for all cases, however, since donor-derived cells from 7 of the 22 patients with
chronic GVHD showed no measurable proliferative response after stimulation with either
recipient or donor cells.
Role of TGF-β. A syndrome characteristic of chronic GVHD develops after
transplantation of B10.D2 lymphoid cells into irradiated BALB/c recipients.22 Skin changes
include a mononuclear infiltrate deep in the dermis, loss of dermal fat, increased collagen
deposition, and “dropout” of dermal appendages such as hair follicles, but in distinction to
findings in acute GVHD, apoptosis of basal epithelial cells at the dermal-epidermal junction
does not occur. Skin changes begin as early as day 11, and cutaneous fibrosis is apparent
as early as day 21. Deposits of IgG, IgA and IgM appear at the dermal epidermal junction
in this model.23 Additional features of the disease in this model include inflammation and
fibrosis in salivary and lacrimal glands, sclerosing cholangitis, progressive renal and
gastrointestinal fibrosis, and development of anti-Scl-70 antibody.24
Naïve donor CD4 cells initiate the disease in this strain combination,25,26 and the
dermal infiltrate is comprised of T cells, monocytes and macrophages.27 Antigen
presenting cells of either the donor or recipient are sufficient to initiate the disease, and
costimulation of donor T cells through CD80 or CD86 on APCs is necessary in order to
induce chronic GVHD.28 In this model, costimulation of donor CD4 cells through CD40 on
APCs is necessary to induce intestinal disease but not skin disease. T cells and
macrophages in the skin express TGF-β1 but not TGF-β2 or TGF-β3 mRNA.29 Microarray
analysis also showed upregulated expression of type 1 (interferon-γ) and type 2 (IL-6, IL-
10 and IL-13) cytokines, chemokines, and a variety of growth factors and cell adhesion
molecules in recipients with chronic GVHD as compared to recipients without chronic
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GVHD.30 Administration of a neutralizing antibody against TGF-β prevented or reduced
virtually all cutaneous manifestations of chronic GVHD, including cellular infiltration,
immune cell activation, thickening and fibrosis.27 In contrast, neutralization of TGF-β by
administration of latency-associated peptide prevented thickening and fibrosis, but did not
prevent influx of T cells and monocytes or immune cell activation in the skin.31
Cutaneous manifestations of chronic GVHD develop after transplantation of B10.D2
spleen cells into MHC-matched (H-2d) BALB/c recipients but not after transplantation of
B10 spleen cells into MHC-matched (H-2b) BALB.B recipients or after transplantation of
B10.BR spleen cells into MHC-matched (H-2k) BALB.K recipients (Table 3).32 In each of
these combinations, the background genes of the donor are of B10 origin, while those of
the recipient are of BALB origin, and the combinations differ from each other only in the
MHC. These observations were interpreted as indicating that H-2d MHC class II molecules
(I-Ad or I-Ed) could present minor antigen peptides derived from BALB skin to CD4 cells of
B10 donors, while H-2b and H-2k class II molecules could not. Another explanation for this
observation could be related to a polymorphism in the TNF-α gene, which is located in the
MHC.33 Expression of TNF-α is reduced in H-2d mice as compared to H-2b or H-2k mice.
Since TNF-α is a potent inhibitor of fibrosis induced by TGF-β, it has been proposed that
the fibrosis observed with B10.D2 donors and BALB/c recipients might be related to an
inability of cutaneous CD4 cells to produce TNF-α.34
Attempts to prevent or treat chronic GVHD through direct or indirect manipulation of
TGF-β may encounter unexpected complexity. For example, Asai et al.35 showed that
activated donor NK cells could attenuate the severity of acute GVHD through a TGF-β-
dependent mechanism. TGF-β has a non-redundant, essential role in limiting T cell and
NK cell responses, and TGF-β-deficient mice develop an early onset lethal autoimmune
disease.36 The ability of TGF-β neutralization to prevent or treat chronic GVHD may
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depend on the context in which the disease develops. Treatment of B10.D2 donors with G-
CSF exacerbates the severity of skin GVHD in sublethally irradiated BALB/c recipients.
Donor T cells are required for the development of GVHD, but the severity of cutaneous
sclerosis is determined by the non-T cell fraction of grafts from G-CSF-treated donors.37 In
this model, neutralization of TGF-β from day 0 – 42 had no effect on manifestations of
GVHD in the skin, liver or gastrointestinal tract, but neutralization of TGF-β beginning on
day 14 appeared to attenuate progression of the disease to some extent in the skin and
gastrointestinal tract, but not the liver.38 Neutralization of TGF-β after transplantation
exacerbated acute GVHD by interfering with the regulatory effects of TGF-β on
proliferation of donor T cells stimulated by recipient alloantigens. Finally, since TGF-β
induces Foxp3 expression and T-regulatory function in CD4+CD25- precursors,39 TGF-β
neutralization could exacerbate GVHD by interfering with development of T regulatory
cells.
The role of TGF-β in human chronic GVHD has not been defined. Results of one study
showed elevated serum levels of TGF-β in patients with chronic GVHD as compared to
patients without chronic GVHD.40 The interpretation of these results is complicated, since
assays were carried out not with plasma, but with serum, which contains large amounts of
TGF-β released from platelets during clotting. Gene expression studies have shown that
increased TGF-β signaling in CD4 cells and CD8 cells was associated with a reduced risk
of chronic GVHD in humans.41 These data might appear to conflict with results from
experiments with B10.D2 donors and BALB/c recipients, but 4 of the 5 TGF-β pathway
genes that were examined also showed increased expression associated with a
decreased risk of acute GVHD. The association of increased TGF-β activity with a reduced
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risk of chronic GVHD might result from a decreased risk of acute GVHD, since acute
GVHD is a well-recognized risk factor for chronic GVHD.
Established chronic GVHD induced by B10.D2 T cells in BALB/c recipients can be
reversed by administration of an agonistic CD137-specific antibody, but administration of
this antibody at the time of the transplant exacerbated the severity of acute GVHD.42
Administration of agonistic CD137-specific antibody has been used successfully to treat
CD4-mediated autoimmune diseases in variety of experimental models. Clinical trials will
be needed to determine the safety and efficacy of this approach for treatment of chronic
GVHD in humans.
Role of B cells. Deposition of antibody at the junction between the dermis and
epidermis has been demonstrated in both murine models23 and in humans8 with chronic
GVHD. Both euthymic and athymic BALB/c recipients developed high levels of double
strand DNA-specific IgG1 and IgG2a autoantibodies in the serum, cutaneous sclerosis,
and glomerulonephritis with proteinuria, beginning within the first 2 weeks after
transplantation of spleen cells from DBA/2 (H-2d) donors.43 Induction of disease required
both donor CD4+CD25- T cells and donor B cells. In the absence of donor B cells, donor
CD4 cells caused acute GVHD without cutaneous sclerosis or glomerulonephritis. The
relevance of this model for human chronic GVHD could be questioned, since double-
strand DNA-specific autoantibodies, immune complex glomerulonephritis and proteinuria
are characteristic of systemic lupus but rarely occur in patients with chronic GVHD.44
Several lines of evidence suggest that B cells are likely to have some role in the
pathogenesis of chronic GVHD in humans. First, anecdotal experience and phase 2
studies have shown clinical improvement in some patients with chronic GVHD after
administration of a CD20-specific antibody.45 Second, biomarker studies have shown
enhanced CD86 expression after TLR9 stimulation of B cells from patients with chronic
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GVHD, as compared to those from controls.46 Third, patients with chronic GVHD have high
levels of B cell activating factor, which promotes the survival and differentiation of
activated B cells.47 Finally, agonistic antibodies against platelet-derived growth factor
receptor (PDGFR) were detected in serum from each of 22 patients with clinical extensive
chronic GVHD but not in serum from any of 17 patients without chronic GVHD.48 These
antibodies induce tyrosine phosphorylation of PDGFR, accumulation of reactive oxygen
species, and stimulation of type 1 collagen gene expression by fibroblasts, through a Ras
and ERK1/2-dependent signaling pathway. These results suggest novel approaches for
treatment of chronic GVHD, since ligand-induced phosphorylation of the PDGFR is
susceptible to inhibition by certain tyrosine-kinase inhibitors, including imatinib.
Role of T regulatory cells. Acute GVHD not only impairs negative selection of T cells in
the thymus, as discussed above, but also impairs development of T regulatory cells,18
which might otherwise prevent the development of chronic GVHD. Anderson et al.49
showed that donor or recipient-derived T regulatory cells could prevent GVHD caused by
B10.D2 CD4 cells in BALB/c recipients, and Zhang et al.43 similarly showed that donor-
derived T regulatory cells could prevent GVHD caused by DBA/2 CD4 cells and B cells in
BALB/c recipients. More impressively, Fujita et al.50 showed that recipient-derived
regulatory dendritic cells cultured in medium containing GM-CSF, IL-10, and TGF-β and
then matured by exposure to lipopolysaccharide were not only able to prevent GVHD
caused by B10.D2 CD4 cells in BALB/c recipients but were also able to reverse previously
established GVHD.
Conflicting results have been reported with respect to the role of T-regulatory cells in
preventing the development of chronic GVHD in humans. Results of at least two studies
suggested that the presence of increased numbers of T regulatory cells in the blood was
associated with a decreased risk of chronic GVHD,51,52 but these results were not
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confirmed by two other studies.53,54 The most persuasive results emerged from a study by
Rieger et al.,55 who showed that colonic mucosa from patients with acute or chronic GVHD
contained very low numbers of T regulatory cells relative to the numbers of CD8 cells,
whereas specimens from patients with cytomegalovirus infection or diverticulitis contained
much higher numbers of T regulatory cells relative to the numbers of CD8 cells.
1B1BFuture prospects
The results summarized above raise more questions than answers and leave the
impression that no animal model fully replicates all of the features of chronic GVHD in
humans, and more importantly, that no single biological mechanism is likely to account for
the diverse features of chronic GVHD in humans. In the future, it might become possible to
tailor treatment for specific abnormalities that contribute to the disease in each individual
patient. The disease is clearly initiated by donor T cells that recognize recipient
alloantigens, since the incidence of chronic GVHD can be decreased by exhaustive
depletion of T cells from the graft. Evidence from animal models has clearly demonstrated
that acute GVHD can cause defects in thymic negative selection, allowing the
development of alloreactive T cells that cause chronic GVHD. Preliminary results have
suggested that administration of KGF could potentially be used to prevent or treat chronic
GVHD that results from this type of mechanism. T cells and B cells can also contribute to
pathogenesis of the disease through mechanisms that do not necessarily depend on prior
acute GVHD or thymic dysfunction. It seems likely that binding of agonistic antibodies to
cell surface molecules other than PDGFR might account for certain manifestations of
chronic GVHD. If so, then a range of small molecule inhibitors of intracellular signal
transduction could one day be used to treat the disease. More importantly, elucidation of
the mechanisms responsible for generating pathogenic autoantibodies could provide a
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basis for development of methods to prevent chronic GVHD. Finally, development of
methods for ex-vivo expansion of T regulatory cells or for improving the generation,
survival and function of these cells in vivo could be used to prevent or reverse chronic
GVHD caused by deficits in the numbers or function of T regulatory cells.
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Acknowledgements
This work was supported by grant CA 98906 from the National Cancer Institute, National
Institutes of Health, U.S. Department of Health and Human Services.
16
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24
Table 1. Overview of Chronic GVHD
• Pleiomorphic syndrome
• Resembles autoimmune diseases
• Onset at 3 – 15 months after transplant
• 40 – 60% incidence among 3-month survivors
• Involvement of multiple organs
• Immune dysfunction with risk of infections
• 30-50% risk of transplant-related mortality
• Reduced risk of recurrent malignancy
25
Table 2. Responses of T Cells from patients*
0 (0)8 (36)By recipient alone, n (%)
0 (0)7 (32)Neither, n (%)1 (8)6 (27)By both, n (%)
0 (0)1 (5)By donor alone, n (%)
No (n = 12)Yes (n = 22)Response after stimulation
Chronic GVHD
*adapted from reference 21
26
Table 3. GVHD phenotypes, according to differences in MHC
TNF-α GVHD Phenotype
Donor Recipient MHC Production Skin Systemic
B10.D2 BALB/c H-2d Low Chronic None
B10.BR BALB.K H-2k High Modified* Present
B10 BALB.B H-2b High None Present
B10 x B10.D2 BALB.B x BALB/c H-2b/d Intermediate Chronic Present
*less severe, especially for hair loss; adapted from reference 32